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Biomedical subjects

M Kew

Publications and source records attributed to M Kew.

At least 19 recordsLinked to original sources
PubMed2006

Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma.

Hepatitis B virus (HBV)-encoded X antigen (HBxAg) contributes to the development of hepatocellular carcinoma (HCC). A frequent characteristic of HCC is reduced or absent expression of the cell adhesion protein, E-cadherin, although it is not known whether HBxAg plays a role. To address this, the levels of E-cadherin were determined in HBxAg-positive and -negative HepG2 cells in culture, and in tumor and surrounding nontumor liver from a panel of HBV carriers. The results showed an inverse relationship between HBxAg and E-cadherin expression both in tissue culture and in vivo. In HBxAg-positive cells, E-cadherin was suppressed at both the mRNA and protein levels. This was associated with hypermethylation of the E-cadherin promoter. Depressed E-cadherin correlated with HBxAg trans-activation function, as did the migration of HepG2 cells in vitro. Decreased expression of E-cadherin was also associated with the accumulation of beta-catenin in the cytoplasm and/or nuclei in tissues and cell lines, which is characteristic of activated beta-catenin. Additional work showed that HBxAg-activated beta-catenin. Together, these results suggest that the HBxAg is associated with decreased expression of E-cadherin, accumulation of beta-catenin in the cytoplasm and nucleus, and increased cell migration, which may contribute importantly to hepatocarcinogenesis.

Cadherins↗
PubMed2004

Prevention of hepatitis C virus infection.

In spite of advances made in our understanding of the biology of the hepatitis C virus (HCV), the epidemiology and natural history of HCV infection, and the treatment of chronic hepatitis C, the development and worldwide implementation of a comprehensive prevention and control strategy remains necessary. A World Health Organization informal consultation with the Viral Hepatitis Prevention Board was convened and met in Geneva, Switzerland, 13-14 May 2002, to review epidemiological and public health aspects of HCV infection, and the various prevention and control strategies that are currently in place. Based on the presentations and discussions, a number of specific recommendations were made, which should be considered in conjunction with previously published recommendations.

Antiviral Agents↗
PubMed2001

Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues.

The hepatitis B virus (HBV) X protein (HBx) is a transcriptional transactivator that has been implicated in the development of HBV-related hepatocellular carcinoma. Mutations in the HBx open reading frame have been reported, but their general impact on the biological function of HBx remains unknown. To address this issue, we comparatively analyzed the structures and biological functions of HBx sequences isolated from sera and from tumor and nontumor tissues of patients with a HBV-related hepatocellular carcinoma. In addition to the HBx sequences derived from free HBV genomes, HBx from HBV integrants was also obtained from the tumor tissues by use of a HBx-Alu PCR-based approach. Sequence analysis showed that the HBx sequences derived from tumor tissues (6 of 7), particularly those isolated from HBV integrants (4 of 4), contained a deletion in the distal COOH-terminal region. Interestingly, most of the COOH-terminally truncated HBx sequences obtained from tumor tissues, in contrast to the full-length HBx isolated from the sera and nontumor tissues, lost their transcriptional activity and their inhibitory effects on cell proliferation and transformation. Importantly, although full-length HBx suppressed the focus formation induced by the cooperation of ras and myc oncogenes in primary rat embryo fibroblasts, COOH-terminally truncated HBx enhanced the transforming ability of ras and myc. Finally, by analyzing the artificial mutants, we were able to more precisely map the functional domains located at the COOH-terminal of HBx. Taken together, our results suggest a key role for the HBx COOH-terminal end in controlling cell proliferation, viability, and transformation. This study further supports the hypothesis that natural HBx mutants might be selected in tumor tissues and play a role in hepatocarcinogenesis by modifying the biological functions of HBx.

Amino Acid Sequence↗
PubMed2001

Increased hepatic oxidative DNA damage in patients with hepatocellular carcinoma.

Since oxidative DNA damage plays a role in experimental carcinogen-induced cancers, the purpose of the present study was to determine if hepatic oxidative DNA damage was increased in patients with HCC compared to patients with benign hepatic tumors or hepatic metastases (non-HCC) or to patients with end-stage alcoholic liver disease undergoing liver transplantation. Oxidative DNA damage was assessed by 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Results showed that peritumoral 8-OH-dG was markedly increased in HCC (N= 51) (180 +/- 74 vs 32 +/- 58-OH-dG/10(6)dG for tumor, P < 0.005) in contrast to patients with non-HCC (N = 17), in whom the peritumoral 8-OH-dG did not differ from that in tumor (39 +/- 7 vs. 31 +/- 108-OH-dG/10(6)dG). Oxidative DNA damage can be both mutagenic and carcinogenic; our data suggested it will be important in future studies to determine the chronology of this type of liver injury relative to hepatocarcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

A cellular gene up-regulated by hepatitis B virus-encoded X antigen promotes hepatocellular growth and survival.

Polymerase chain reaction (PCR) select complementary DNA (cDNA) subtraction of hepatitis B x antigen (HBxAg)-positive compared with -negative HepG2 cells resulted in the up-regulated expression of a cellular gene that encodes a transcript of 745 bases and a polypeptide 99 amino acids long. GenBank analysis revealed extensive homology with the amino terminal domain of cellular multidrug resistant proteins (MRP), although overexpression of this gene did not confer an MRP phenotype. In situ hybridization and immunostaining showed colocalized expression with HBxAg in the liver of hepatitis B carriers. Overexpression of this protein stimulated the growth of HepG2 cells in serum-free medium, and partially protected cells from anti-Fas-mediated killing, but did not promote growth in soft agar or tumor formation in nude mice. Introduction of the dominant negative inhibitor of nuclear factor kappaB (IkappaBalpha) into HBxAg-positive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, suggesting that its up-regulation is nuclear factor kappaB (NF-kappaB) dependent. Hence, HBxAg activation of NF-kappaB may result in the up-regulation of a cellular protein that promotes growth factor-independent survival and protects against Fas-mediated killing. This factor may contribute to the persistence of infected hepatocytes during chronic infection, which is important for the later development of hepatocellular carcinoma (HCC).

ATP-Binding Cassette Transporters↗
PubMed2001

Hepatitis B virus and hepatocellular carcinoma.

Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC.

Animals↗
PubMed2000

In situ demonstration of inhibitory effects of hammerhead ribozymes that are targeted to the hepatitis Bx sequence in cultured cells.

Chronic hepatitis B virus (HBV) infection is endemic to several populous areas of the world and is frequently complicated by hepatocellular carcinoma. Ribozymes can be designed to cleave target RNA sequences specifically and show promise for the treatment of HBV infection. Demonstration of intracellular inhibition of HBV gene expression, essential to developing therapeutic ribozymes, has been the aim of this investigation. We generated two vectors encoding hammerhead ribozymes that target the HBx region of HBV. Plasmids containing intact HBV sequences or a modification in which the preS2/S region was replaced by DNA encoding enhanced green fluorescent protein (EGFP) were used to test ribozyme action in transfected cells. Both ribozymes inhibited surface antigen secretion and EGFP expression similarly. The measurement of EGFP expression is convenient to assess ribozyme action in situ and effective targeting of HBV sequences that are common to all HBV transcripts is potentially useful to develop strategies to counter HBV infection.

Base Sequence↗
PubMed2000

Hammerhead ribozyme-mediated inhibition of hepatitis B virus X gene expression in cultured cells.

BACKGROUND/AIMS: Chronic infection with hepatitis B virus (HBV) is endemic to sub-Saharan Africa and parts of Asia. Common complications of HBV persistence include cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important objective. The HBV X protein (HBx) is a transcriptional activator that is required for the establishment of HBV infection and is implicated in hepatocarcinogenesis. The aim of this study was to assess the ability of two endogenously expressed hammerhead ribozymes to inhibit expression of HBV genes in transfected cultured cells. METHODS: Eukaryotic expression plasmids producing two ribozymes targeted to the HBx open reading frame, as well as their catalytically inactive homologues, were generated. Established cell lines and a primary culture of malignant hepatocytes were transfected to assess ribozyme effects on HBx expression and HBV replication. RESULTS: The ribozyme-expressing vectors inhibit expression of functional HBx protein and decrease HBV mRNA encoding surface and HBx sequences in transfected cells. Moreover, decreased HBsAg and HBeAg secretion from cells transfected with the ribozymes and an HBV replication competent plasmid provide evidence for an antireplicative effect of the ribozymes. However, the data do not exclude a dominant antisense effect that inhibits HBV gene expression. CONCLUSIONS: Inactivation of HBx, a sequence that is conserved in mammalian hepadnaviruses and found in all HBV transcripts, has potential for the treatment of chronic HBV infection.

Base Sequence↗
PubMed2000

Putative role of hepatitis B virus X protein in hepatocarcinogenesis: effects on apoptosis, DNA repair, mitogen-activated protein kinase and JAK/STAT pathways.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV-induced malignant transformation is, however, incompletely understood. HBx, the protein encoded by the X open reading frame, is a transcriptional activator that has been implicated in hepatocarcinogenesis. HBx inhibits the function of the tumour suppressor protein p53 in what is thought to be an early event in hepatocyte transformation before the later accumulation of inactivating p53 point mutations. HBx inhibits apoptosis but also exerts pro-apoptotic effects. The effects of HBx on apoptosis may be important not only for the development of HCC but also for the establishment of HBV infection. Further implication of HBx in hepatocyte transformation has been the demonstration that it inhibits the repair of damaged hepatocyte DNA. This effect may be mediated by interaction with p53 or through binding to the damaged DNA binding protein (DDB), which plays an accessory role in nucleotide excision repair. In addition, HBx activates cell signalling cascades involving mitogen-activated protein kinase (MAPK) and Janus family tyrosine kinases (JAK)/signal transducer and activators of transcription (STAT) pathways. The implications of these modulating effects of HBx are not fully understood, but they are likely to have wide-ranging effects on hepatocyte proliferation, apoptosis and the regulation of cell growth checkpoints. The cellular functions ascribed to HBx are unusually diverse, and defining the biologically important role of HBx during HBV replication will go some way to understanding the sequelae of chronic HBV infection.

Animals↗
PubMed1999

Smad2 and Smad4 gene mutations in hepatocellular carcinoma.

TGF-beta is a negative regulator of liver growth. Smad family of genes, as mediators of TGF-beta pathway, are candidate tumor suppressor genes in hepatocellular carcinoma (HCC). We studied 35 HCC and non-tumour liver tissues for possible mutations in Smad2 and Smad4 genes. Three tumours displayed somatic mutations; two in Smad4 (Asp332Gly and Cys401Arg) and one in Smad2 (Gln407Arg) genes. All three mutations were A:T --> G:C transitions suspected to result from oxidative stress as observed in mitochondrial DNA. These observation demonstrate that TGF-beta pathway is altered in hepatocellular carcinoma.

Base Sequence↗
PubMed1999

The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis.

The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the differences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One differentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma. Introduction of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice. Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of hepatocellular carcinoma.

Amino Acid Sequence↗
PubMed1996

Allele-specific PCR analysis of p53 codon 249 AGT transversion in liver tissues from patients with viral hepatitis.

AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas where exposure to aflatoxin B1 (AFB) occurs. We developed a sensitive allele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from the wild-type allele due to a G-to-T transversion in its terminal 3' nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 105 copies of the wild-type allele and was 100-fold more sensitive than the restriction fragment length polymorphism-PCR technique. We have applied this AS-PCR protocol to examine codon 249 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non-neoplastic liver from Mozambiquan patients, all of whom were HBsAg- or HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neoplastic liver from North American patients with either HBV- or HCV-derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non-neoplastic hepatocyte populations.

Adult↗
PubMed1996

Precore/core mutations of hepatitis B virus in hepatocellular carcinomas developed on noncirrhotic livers.

BACKGROUND & AIMS: Hepatitis B virus genomes have been detected in hepatocellular carcinoma developed without the pretumoral step of cirrhosis; this finding is consistent with a direct effect of the virus in liver cell transformation. The aim of this study was to investigate the molecular bases for the hepatitis B virus persistence. Three hepatitis B surface antigen-positive and 2 hepatitis B surface antigen-negative patients with hepatocellular carcinoma and without cirrhosis were studied for precore/core mutations in tumorous and nontumorous tissues. METHODS: The precore/core region was amplified and analyzed both by cloning and sequencing and by direct sequencing. RESULTS: Nonsynonymous mutations were identified selectively in tumorous tissues. Clusters of mutations were identified in the viral encapsidation (epsilon) signal, in immunodominant epitopes of the core protein, and in the polymerase. Common associated point mutations were found in 3 patients infected with hepatitis B virus genotype A in the epsilon signal and in the polymerase gene. CONCLUSIONS: Precore/core mutations located in domains involved in hepatitis B virus replication and immune response to the virus were identified. The selective presence of some of these mutations in tumorous tissues is consistent with their role in the persistence of the virus in neoplastic cells and, possibly, in liver oncogenesis.

Adult↗
PubMed1995

Selective accumulation of the X transcript of hepatitis B virus in patients negative for hepatitis B surface antigen with hepatocellular carcinoma.

In HBsAg-negative patients with hepatocellular carcinoma (HCC), hepatitis B virus (HBV) genomes are present at a low copy number per cell, and the role of HBV in liver transformation is still unclear. We have mapped by polymerase chain reaction (PCR) the HBV genome in 19 HBsAg-negative tumorous and 9 corresponding nontumorous tissues and evaluated, by RT-PCR, the presence of HBV S, X, and C transcripts in the tumorous and nontumorous tissue of nine HBsAg-negative and, for comparison, six HBsAg-positive patients. Disrupted, presumably integrated, HBV genomes were detected by PCR in 10 of 19 tumorous tissues and in only one of nine nontumorous tissues. Significant accumulation of viral RNAs containing X but not C or S sequences was shown in 7/9 tumors and 7/8 nontumorous tissues from HBsAg-negative patients. In contrast, viral RNAs revealed by X-as well as by S- and C-specific primers were detected in five of six tumors and in six of six nontumorous tissues from HBsAg-positive patients. In conclusion, our results suggest the frequent integration of the HBV genome and the accumulation of X-related RNAs in HCCs developing in HBsAg-negative patients. This finding is consistent with a role, in these cases, for the potentially transforming X protein.

Adolescent↗
PubMed1994

Genetic heterogeneity of hepatocellular carcinoma.

We studied 80 hepatocellular carcinomas from three continents for p53 gene (TP53) mutations and hepatitis B virus (HBV) sequences. p53 mutations were frequent in tumors from Mozambique but not in tumors from South Africa, China, and Germany. Independent of geographic origin, most tumors were positive for HBV sequences. X gene coding sequences of HBV were detected in 78% of tumors, whereas viral sequences in the surface antigen- and core antigen-encoding regions were present in less than 45% of tumors. These observations indicate that hepatocellular carcinomas are genetically heterogeneous. Mozambican-type of hepatocellular carcinomas are characterized by a high incidence of p53 mutations related to aflatoxins. In other tumors, the rarity of p53 mutations combined with the frequent presence of viral X gene coding sequences suggests a possible interference of HBV with the wild-type p53 function.

Base Sequence↗
PubMed1991

Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa.

Hepatocellular carcinoma (HCC) is a prevalent cancer in sub-Saharan Africa and eastern Asia. Hepatitis B virus and aflatoxins are risk factors for HCC, but the molecular mechanism of human hepatocellular carcinogenesis is largely unknown. Abnormalities in the structure and expression of the tumour-suppressor gene p53 are frequent in HCC cell lines, and allelic losses from chromosome 17p have been found in HCCs from China and Japan. Here we report on allelic deletions from chromosome 17p and mutations of the p53 gene found in 50% of primary HCCs from southern Africa. Four of five mutations detected were G----T substitutions, with clustering at codon 249. This mutation specificity could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1 (ref. 7), a food contaminant in Africa, which is both a mutagen that induces G to T substitution and a liver-specific carcinogen.

Aflatoxins↗